Knock-in non-human animal producing human tissue factor

ABSTRACT

A non-human animal that produces human tissue factor (TF) without substantially producing non-human animal tissue factor, said animal having a genome in which cDNA encoding human TF has been inserted upstream of the translation initiation codon for the non-human animal genomic TF gene.

FIELD OF THE INVENTION

The present invention relates to knock-in non-human animals that producehuman tissue factor (hTF).

BACKGROUND ART

Inhibiting agents of tissue factor (TF) are useful as inhibitors ofblood coagulation, inhibitors of angiogenesis, therapeutic agents forarteriosclerosis, and the like. However, inhibiting agents of humantissue factor are highly species-specific, and, for example, anti-humanTF antibody does not react to tissue factors from animals other thanprimates. Therefore, the creation of animals that express human TF ishighly desired for the evaluation of inhibiting agents of human TF.

Though a knock-out mouse for human TF exhibits embryonic lethality, theexpression of human TF can circumvent embryonic lethality (Parry G C Net al., J. Clin. Invest. 101:560-569 (1998)). However, as this mouse hasan introduced human TF minigene, the expression of tissue factor is lowand thus breeding performance is poor (Erlich J. et al., Proc. Natl.Acad. Sci. USA 96:8138-8143 (1999)).

DISCLOSURE OF THE INVENTION

Thus, the present invention is intended to provide a non-human animalthat can produce human TF.

In order to solve the above problem, the present invention provides anon-human animal that produces human TF without substantially producingnon-human animal tissue factor. More specifically, the present inventionprovides a non-human animal which has a genome wherein a humanTF-encoding gene has been inserted. Preferably, the human TF-encodinggene has been inserted onto the same chromosome as that for non-humananimal TF gene. Preferably, the human TF-encoding gene has been insertedupstream of the translation initiation codon for non-human animal TFgene. Preferably, the human TF-encoding gene comprises an AU-richresponse element (ARE) or a polyA additional signal at the 3′-endthereof. More preferably, the human TF-encoding gene comprises anAU-rich response element (ARE) and a polyA additional signal at the3′-end thereof. Preferably, the non-human animal is a rodent, forexample a mouse.

The present invention also provides various methods for using theabove-mentioned knock-in non-human animal.

Thus, the present invention provides a method of screening a therapeuticagent for a disease caused by human TF, said method comprising:

(a) administering a test substance to a knock-in non-human animal thatproduces the human TF of the present invention or a non-human animalobtained by mating said non-human animal with another non-human animal;and

(b) determining whether or not the symptom of the disease caused byhuman TF has been suppressed in the non-human animal that received saidtest substance.

For example, said disease caused by human TF is thrombosis or sepsis.

The present invention also provides a method of confirming whether ornot a test substance is safe, said method comprising:

(a) administering a test substance to a knock-in non-human animal thatproduces the human TF of the present invention or a non-human animalobtained by mating said non-human animal with another non-human animal;and

(b) confirming whether or not the non-human animal that received saidtest substance develops a bleeding symptom.

The present invention also provides a method of screening an anti-tumoragent, said method comprising:

(a) administering a test substance to a knock-in non-human animal thatproduces the human TF of the present invention or a non-human animalobtained by mating said non-human animal with another non-human animal;and

(c) determining whether or not tumor has been suppressed in thenon-human animal that received said test substance.

The present invention also provides a method of screening anangiogenesis-inhibiting agent, said method comprising:

(a) administering a test substance to a knock-in non-human animal thatproduces the human TF of the present invention or a non-human animalobtained by mating said non-human animal with another non-human animal;and

(c) determining whether or not angiogenesis has been suppressed in thenon-human animal that received said test substance.

The present invention also provides a method of screening a therapeuticagent for arteriosclerosis, said method comprising:

(a) administering a test substance to a knock-in non-human animal thatproduces the human TF of the present invention or a non-human animalobtained by mating said non-human animal with another non-human animal;and

(b) determining whether or not arteriosclerosis has been suppressed inthe non-human animal that received said test substance.

The present invention also provides a method of preparing anti-TFantibody against non-human animal TF, said method comprising immunizingthe above non-human animal with tissue factor derived from saidnon-human animal.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a drawing that schematically illustrates the formation ofgenomic DNA in the creation of a human tissue factor (TF) knock-inmouse. In the figure, A schematically illustrates the relation of amouse TF genomic DNA (1) and a full-length human TF cDNA and theneomycin-resistant gene to be inserted (2). B is a drawing thatschematically illustrates how a mouse TF genomic DNA (a) and a knock-invector (b) undergo homologous recombination to form a knock-in genomicDNA (c), wherein the x mark between (a) and (b) indicates thathomologous recombination takes place between (a) and (b).

FIG. 2 is a drawing showing the result of Northern blot analysis thatindicates the tissue distribution of TF gene expression in the mouse.

FIG. 3 is a drawing showing the result of Northern blot analysis thatindicates the expression products of various mice related to the presentinvention.

Referring to FIG. 4, the expression of TF in the brain of a wild-typemouse and a knock-in mouse was detected by (A) anti-mouse TF antibodyMFR-37 and (B) anti-human TF antibody ADI#4503, and they are photographsshowing the result of Western blot analysis.

FIG. 5 is a graph showing a result in which the inhibition of mouseplasma coagulation by anti-human TF antibody in a wild type mouse and ahuman TF knock-in mouse was determined by measuring plasma coagulationtime, wherein the y-axis represents the plasma coagulation time and eachcolumn represents the final concentration of antibody.

FIG. 6 is a graph similar to FIG. 5 but shows the result ofdetermination of human plasma coagulation time.

FIG. 7 is a graph showing a result of generation of anti-mouse TFantibody in Working Example 5, wherein the y-axis represents absorbanceat 405 nm (reference 655 nm) and the x-axis represents the serumdilution factor. It shows the result of the 07 line mouse No. 1.

FIG. 8 is a graph similar to FIG. 7 showing the result of the 07 linemouse No. 2.

FIG. 9 is a graph similar to FIG. 7 showing the result of the 07 linemouse No. 3.

FIG. 10 is a graph similar to FIG. 7 showing the result of the 76 linemouse No. 4.

FIG. 11 is a graph similar to FIG. 7 showing the result of the 76 linemouse No. 5.

FIG. 12 is a graph similar to FIG. 7 showing the result of the 76 linemouse No. 6.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to produce human TF in non-human animals, a gene encoding humanTF may be inserted into a suitable site in the genome of the non-humananimals (transgenic non-human animals). However, if it is desired toobtain non-human animals that satisfy the two requirements of notproducing non-human animal TF and of producing human TF, it is necessaryto inactivate the native TF gene of the non-human animals. On the otherhand, since it is known from Parry et al., J. Clin. Invest. 101:560(1998) that there is a regulatory element for expression in intron 1between exon 1 and exon 2 of TF, the non-human animal TF genomic genewas not deleted, but the method of inactivating the expression of thenon-human animal TF gene was adopted.

Thus, according to the present invention, as a site into which the humanTF-encoding gene is to be inserted, the site on the same chromosome asthat for the genomic gene of the non-human animal TF, specificallyupstream of the translation initiation codon of the genomic gene of thenon-human animal TF is preferable. By this approach, it is expected thathuman TF gene only is expressed and no TF gene of the non-human animalsis expressed based on a biological rule that the first initiation codonis preferentially used when a plurality of initiation codons occur inseries. Furthermore, the presence of a polyA additional signal at the3′-end of human TF cDNA would prevent read through of the genomic geneof the non-human animal TF.

Human TF of the present invention may be the human TF having thefull-length amino acid sequence or fragments (preferably peptides of 100or more amino acid residues, and more preferably peptides of 200 or moreamino acid residues, and for example a peptide containing 274 aminoacids of amino acid residues −32 to 242 as described in Morrissey etal., Cell 50:129-135 (1987)) of human TF, and preferably human TF havingthe full-length amino acid sequence. Thus, the gene encoding human TF isnot specifically limited, and it may be a gene encoding the full-lengthhuman TF or a gene encoding a fragment thereof, and it may preferably bea gene encoding the full-length human TF.

The gene encoding human TF of the present invention is not specificallylimited as long as it is a gene capable of expressing human TF and, forexample, cDNA or genomic DNA may be used. According to the presentinvention, the preferred gene encoding human TF is cDNA. Furthermore,other genes such as regulatory genes that regulate the expression ofgene encoding human TF of the present invention may be added to the saidgene. The origin of regulatory genes that are added to human TF is notspecifically limited, and the genes may be derived from humans or fromnon-human animals.

Furthermore, according to the present invention, an AU-rich responseelement (ARE), a polyA additional signal, an untranslated region (UTR)etc. may be added to the gene encoding human TF. As the AU-rich responseelement (ARE), sequences known to a person skilled in the art may beused, and for example ATTTA etc. may be used. As the polyA additionalsignal, sequences known to a person skilled in the art may be used, andfor example AATAAA etc. may be used. As the untranslated region, therecan be mentioned for example 3′ UTR and 5′ UTR, with 3′ UTR beingpreferred. The origin of an AU-rich response element (ARE), a polyAadditional signal, an untranslated region (UTR) etc. for use in thepresent invention is not specifically limited, and those derived fromhumans and from non-human animals may be used.

When an AU-rich response element (ARE), a polyA additional signal, anuntranslated region or the like is added to the gene encoding human TF,one of them may be added, and preferably two or more of them are added(for example, an AU-rich response element (ARE) and a polyA additionalsignal, or an untranslated region and a polyA additional signal, etc.).Alternatively, when an AU-rich response element (ARE), a polyAadditional signal, an untranslated region or the like is added, aplurality of the same species may be added (for example, when polyAadditional signals are added, two or more of polyA additional signals ofthe same sequence or of different sequences may be added). Then, avector containing the above structure may be introduced into ES cells ina standard method, and the ES cells are injected into non-human animalembryos to reproduce the non-human animal.

If the non-human animal TF is not substantially reproduced, it meansthat the expression of the non-human animal TF has been artificiallysuppressed and, preferably, the non-human animal TF cannot be detected.As methods of detecting non-human animal TF, for example, a detectionmethod using the anti-TF antibody against non-human animal TF describedin Example 2 may be mentioned.

The non-human animals of the present invention are not specificallylimited, and rodents such as mice, rats and hamsters, non-human primatessuch as monkeys and chimpanzees, mammals such as sheep, cattle and pigs,avians, amphibians, reptiles, fish and the like may be used, andpreferably rodents, and more preferably mice.

The knock-in non-human animals of the present invention can be used asanimal models for diseases caused by human TF. Diseases caused by humanTF include, for example, thrombosis (for example, disseminatedintravascular coagulation syndrome; DIC), arterial stenosis andocclusion, cerebral infarction, venous thrombosis, pulmonary embolism,intraatrial thrombosis), sepsis caused by infection, etc. Furthermore,the knock-in non-human animals of the present invention can be used forthe creation of disease model animals such as tumors, angiogenesis andarteriosclerosis in addition to the above diseases.

Thus, by using the knock-in non-human animals of the present inventionas these disease models, therapeutic agents or preventive agents forthese diseases can be screened.

For example, screening of therapeutic agents or preventive agents fordiseases caused by human TF may be carried out by administering a testsubstance to a knock-in non-human animal of the present invention inwhich thrombosis was induced, and then determining whether or not thedisease caused by human TF has been suppressed.

Furthermore, screening can also be done using non-human animals of thepresent invention that have been fed with a chow. For example, non-humananimals obtained after a high fat diet may be used to screen drugs forarteriosclerosis. Furthermore, non-human animals obtained aftertransplanting a tumor to the non-human animals of the present inventionmay be used to screen anti-tumor agents or angiogenesis inhibitors.

In the screening method for angiogenesis inhibitors, non-human animalsobtained by administering angiogenesis-inducing agents, such as FGF,VEGF and PDGF known to those skilled in the art, to the non-humananimals of the present invention can be used.

In the screening method for therapeutic agents or preventive agents forsepsis, non-human animals can also be used in which sepsis was inducedby administering bacteria or bacterial components to the non-humananimals of the present invention or by extruding bacteria etc. occurringin the intestine of the non-human animals out of the intestine using asurgical method. As bacteria, there can be mentioned, for example, Gramnegative bacteria such as Neisseria meningitidis and Pseudomonasaeruginosa etc. and Gram positive bacteria such as staphylococci andListeria, but Gram negative bacteria are preferred. As preferredexamples of bacterial components, there can be mentioned endotoxinspresent as a component of bacterial cell wall, and lipopolysaccharide(LPS), a component of endotoxins, may be used alone.

Furthermore, screening may be carried out using non-human animalsobtained by crossing the knock-in non-human animals of the presentinvention with other non-human animals. Thus, according to the presentinvention, not only the cases that used the non-human animals of thepresent invention but also the cases that used non-human animalsobtained by crossing the non-human animals of the present invention withother non-human animals are included in the screening method using thenon-human animals of the present invention.

For example, in the case of mice, screening of anti-tumor agents oranti-angiogenesis inhibitors can be carried out using mice obtained bycrossing the knock-in mice of the present invention with cancer-pronemice. As cancer-prone mice, those known to those skilled in the art canbe used, and for example C3H mice, AKR mice, 129 mice and the like canbe used. Furthermore, it is also possible to transplant a tumor to miceobtained by crossing with Severe Combined Immunodeficiency Disease(SCID) mice (Bosma G C et al., Nature 301:527-530 (1983);

available from CLEA Japan Inc.) or with nude mice and then to screenanti-tumor agents or anti-angiogenesis inhibitors.

Furthermore, it is also possible to screen anti-arteriosclerosis agentsby using mice obtained by crossing the knock-in mice of the presentinvention with arteriosclerosis-prone mice (for example, apolipoproteinE (ApoE) knock-out mice (Piedrahita J A et al., Proc. Natl. Acad. Sci.USA 89:4471-4475 (1992); available from The Jackson Laboratory (USA),LDLR knock-out mice). Even when screening is carried out using miceobtained as described above, it is included in the screening methodusing mice of the present invention.

Test substances for use in screening are not specifically limited, andthere can be mentioned, for example, peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cell extracts andthe like.

With respect to the screening method of the present invention, inaddition to the knock-in non-human animals of the present invention andnon-human animals obtained by crossing said knock-in non-human animalswith other non-human animals, biological samples such as organs,tissues, cells, blood and the like harvested from these non-humananimals may be used.

Whether or not diseases caused by human TF are suppressed can be judged,when the disease is disseminated intravascular coagulation syndrome(DIC), using as an index the Ministry of Health and Welfare DICdiagnostic criteria (revised in 1988), or the DIC scoring system (TaylorF B, Jr. et al., (2001) Thromb. Haemost. 86:1327-1330) etc. In the caseof arterial stenosis and occlusion, it can be judged by the patency rateof the blood vessel or the presence or absence of restenosis; in thecase of cerebral infarction, it can be judged by whether or not theformation of cerebral infarction foci has been suppressed in the middlecerebral artery occlusion model; in the case of sepsis, it can be judgedby whether or not a mortality rate has been improved; in the case ofvenous thrombosis, it can be judged by whether or not the amount ofthrombi has been reduced; in the case of pulmonary embolism, it can bejudged by whether or not a mortality rate has been improved; in the caseof intraatrial thrombosis, it can be judged by whether or not the amountof thrombi has been reduced.

Also, whether or not tumor growth or angiogenesis has been suppressedcan be judged by the size or frequency of their formation, and whetheror not arteriosclerosis has been suppressed can be judged on whether ornot hyperplasia of neointima has been suppressed after non-invasiveintravascular treatment.

As the non-human animals of the present invention have a characteristicsthat they do not produce non-human animal TF, they can be used for thepreparation of anti-TF antibody against non-human animal TF. Anti-TFantibody against non-human animal TF can be prepared by immunizing theknock-in non-human animals of the present invention with non-humananimal TF purified from the brain etc. of normal non-human animals, orrecombinant non-human animal TF prepared by using hosts such asEscherichia coli, yeast, insect cells, mammalian cells, or partialpeptide fragments of non-human animal TF (Declerck P J et al. (1995) J.Biol. Chem. 270:8397-8400: an example of method of preparing antibody byimmunization into knock-out animals), or by injecting naked DNA thatexpresses non-human animal TF into the non-human animals of the presentinvention. Generation of antibody can be carried out according to aknown standard method, in which polyclonal antibody (antiserum) ormonoclonal antibody can be prepared.

Also, the non-human animal of the present invention can be used for theevaluation of safety (for example, evaluation of bleeding symptoms) etc.when therapeutic agents (for example, anti-human TF antibody orsubstances that bind to human TF), for diseases caused by human TF, wereadministered.

Furthermore, biological samples such as organs, tissues, cells and bloodharvested from the non-human animals of the present invention may beused to prepare diagnostics or reagents derived from human TF. Thesediagnostics or reagents can be used not only for the diagnosis ofdiseases caused by human TF but also for the evaluation of therapeuticagents. For example, they may be used as reagents for determiningprothrombin time by replacing human-derived thromboplastin reagents.

Now, the present invention will be explained more specifically below.

EXAMPLE 1 Establishment of Knock-In Mice

(1) Construction of Vectors

The mouse TF (mTF) genomic gene was cloned by a PCR method withreference to a report by Mackman et al. (Arteriosclerosis and Thrombosis12:474, 1992). An about 3 kb region of mTF from exon 1 to exon 2 wasamplified with a primer set of mTF1SpS and mTF2A, and then cloned intothe pGEM-T-Easy vector (Promega). The sequences of the primers are asfollows: mTF1SpS: 5′-CGA GCA AAT GCT ACT AGT AGG ATA AGT GAT CGT CTA AGGC-3′ (SEQ ID No. 1); MTF2A: 5′-CTG TAC AGT GTA GGT ATA GTT GGT GGG TTTGGG TTG-3′ (SEQ ID No. 2).

The composition of the reaction mixture for the PCR method was: mousegenomic DNA (derived from ES cells, the 129 mice, 400 μg/ml), 1 μl;10×LA buffer, 5 μl; dNTP, 8 μl; mTF1S pS (100 μM), 0.1 μl; mTF2A (100μM), 0.1 μl; distilled water, 35.3 μl; LA Taq (TAKARA) enzyme, 0.5 μl.PCR comprised a preheating at 94° C. for 1 min, 30 cycles ofamplification reaction of 98° C. for 20 seconds and 68° C. for 15minutes, as well as heating at 72° C. for 10 minutes.

Also, an about 10.5 kb region from exon 2 to exon 6 of the mTF gene wascloned by a PCR method. The primer set used is as follows and the PCRcondition was the same as above. mTF1SpS: 5′-CGA GCA AAT GCT ACT AGT AGGATA AGT GAT CGT CTA AGG C-3′ (SEQ ID No. 1); mTFe6ScA: 5′-ATC AGA GCTCTC CGC AAC AGT GCC GT-3′ (SEQ ID No. 3). The fragment amplified wascleaved with a SacI restriction enzyme and cloned into pBluescript(STRATAGENE).

The 3′-UTR (regulatory region for expression) of human TF (hTF) cDNA wascloned from mRNA of J82 cells (human bladder cancer) using the followingprimers and BcaBEST RNA PCR kit (TAKARA). ShTF3: 5′-TGT TCA AGC AGT GATTCC C-3′ (SEQ ID No. 4); RhTF2: 5′-AAC AAT TCC CAG TCA CCT T-3′ (SEQ IDNo. 5).

The cloned 1.37 kb fragment, after confirming the sequence, was ligatedto the coding sequence (CDS) of hTF at the HpaI site to construct thefull-length hTF cDNA. Though the periphery of the initiation codon ofthe full-length hTF cDNA has been converted to the Kozak sequence, 3′UTR region, which is based on a report by Mackman et al. (Biochemistry28:1755, 1989), includes a region up to the site where polyA has beenadded (AATAAAGGTGACTGGGAATTGTT, the underlined part represents a polyAadditional signal) (SEQ ID NO: 6).

The sequence is shown in SEQ ID NO: 7. In the sequence, bases No. 1-11have been altered to the Kozak sequence (Nucleic Acids Research 15:8125,1987), ATG of bases 12-14 is the translation initiation codon for humanTF, and the coding region of human TF ends at base 896.

Using these mTF genomic DNA and the full-length hTF cDNA, a knock-invector shown in FIG. 1 was constructed. Human TF (hTF) cDNA was insertedinto the Kpn I site preceding the initiation codon (ATG) in exon 1 ofmTF together with a drug resistance gene (neo). As Parry et al. havereported the presence of a regulatory element for expression in theintron (between exon 1 and exon 2) of the TF gene (J. Clin. Invest.101:560, 1998), the procedure of deleting the genome of mTF was notcarried out.

However, as this vector has hTF cDNA preceding the mTF gene and also hasa polyA additional signal, this knock-in mouse was expected to expressonly the hTF gene. It was believed that even if hTF was read through,without added polyA, to transcribe the mTF gene, hTF is only translatedfrom the fusion mRNA and mTF protein would not be produced based on abiological rule that the first initiation codon antecedes in thetranslation of protein.

Furthermore, since this vector has exon 2 of mTF in parallel in theconstruct, it was thought that, even if the protein of mTF istranslated, it would not be functional. The gene of diphtheria toxin Afragment (DTA) is a negative selection marker to obtain homologousrecombinant efficiently. Also, as the promoter of the pgk gene has beenadded to a drug resistance gene, neo, the neo gene is expressed in EScells, and this portion suppresses the expression of the hTF gene thatwas introduced upstream, a procedure of extracting this portion (neo)was added. For this purpose, the neo gene was flanked by the loxPsequence (ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA T) (SEQ ID NO: 8),providing a mechanism such that, when Cre acts thereon, the resultingrecombination will extract the intercalated neo gene.

(2) Introduction into ES Cells

Using the Mouse Kit (LEXIION GENETICS Inc.) commercially available fromTAKARA, the above hTF knock-in (KI) vector was electroporated into EScells (AB2.2 cells derived from 129 SvEv mice), and the homologousrecombinants were screened by a PCR method. The vector (60 μg) waslinearized with Not I, extracted with phenol/chloroform, precipitatedwith ethanol, and then dissolved in PBS for use.

ES cells used in screening were cultured in a 96-well plate, washedtwice in 200 μl per well of the PBS solution, and then a cell lysisbuffer of the following composition (10×LA buffer (for TAKARA LA Taq), 5μl; 5% NP-40, 5 μl; proteinase K (TAKARA, 20 mg/ml), 4 μl; distilledwater, 36 μl) was added thereto, which was treated at 55° C. for 2hours, followed by a treatment at 95° C. for 15 minutes to inactivateproteinase K to provide a PCR sample.

The PCR reaction mixture comprised: the sample, 1 μl; 2×GC buffer, 25μl; dNTP (2.5 mM), 8 μl; primer (20 μM each), 1 μl each; LA Taq(TAKARA), 0.5 μl; and distilled water, 13.5 μl (a total of 50 μl). ThePCR condition comprised a preheating at 95° C. for 1 min, 40 cycles ofamplification reaction of 95° C. for 30 seconds and 64° C. for 30seconds and 72° C. for 1 minute 20 seconds, as well as heating at 72° C.for 10 minutes.

Primers are as described below. In samples of ES cells that underwenthomologous recombination, an about 1.1 kb band is amplified. Primer mTF2was placed in the mTF genomic region of the 5′-end outside of the KIvector, and hTF-A was placed inside of hTF cDNA (see FIG. 1). mTF2(forward): 5′-CCA GTA GGA TAA GTG ATC GTC TAA GGC-3′ (SEQ ID No. 9);hTF-A (reverse): 5′-GCC ACA GTA TTT GTA GTG CCT GAA GC-3′ (SEQ ID No.10).

The result of screening (efficiency of homologous recombination) isshown below. In the experiment, in two runs, 1,180 ES cells wereanalyzed to obtain a total of 13 clones that underwent homologousrecombination (the efficiency of recombination was 1.1% on the averageof two runs).

TABLE 1 No. of ES cells No. of homologous Efficiency of Run No. analyzedrecombinants recombination 1 484 8 1.70% 2 696 5 0.70% Total 1180 131.10%

(3) Establishment of Knock-In Mice

Homologous recombinant ES clones were suspended by trypsin treatment,and washed in the ES cell medium. At an interval of 48 hours, 5 IU ofequine chorionic gonadotropin (eCG) and human chorionic gonadotropin(hCG) were intraperitoneally given to c57BL/6J female mice to inducesuperovulation, then they were mated with c57BL/6J male mice. The daywhen the plug of female mice was confirmed was regarded as day 0.5. Ongestation day 2.5, the uterus and the oviduct were flushed, and themorula stage and 8 cell stage embryos were collected. The collectedembryos were cultured overnight at 37° C. and were developed to theblastocyst stage. 10-15 ES cells were injected into these blastocystes.The embryos after injection were transplanted into the uterus of thepseudoprognancy recipient mice of the ICR at a gestation day 2.5, andthe offspring were obtained 17 days later.

The following table is a summary of the result of injection. It is wellknown that the chimeras with a high proportion of ES-cell-derived coatcolor (having a high percentage of agouti color) are contributing togerm line. And if the XY (ES cells) contribution is high, the chimeraswill typically develop as males. Thus, ES clones 7, 76, and 80 areexpected to be mice capable of transmitting the KI allele.

TABLE 2 No. of No. of No. of No. of hair transferred implantedoffsprings color chimera Proportion of ES cell-derived coat Cloneembryos embryos (%) Total (%) ♂ ♀ Total (%) ♂ ♀ color on male chimeras75 48 22 46% 16 33% 10  6 2 13% 2 0  60, 20% 7 94 73 78% 31 33% 22  913  42% 10  3  90, 90, 90, 90, 90, 90, 70, 70, 70%  1 32 22 69%  2  6% 11 0  0% 0 0 18 32 24 75% 11 34% 4 7 4 36% 1 3  60% 76 46 29 63% 11 24% 74 3 27% 3 0 100%, 95%, 20% 28 34 17 50% 10 29% 4 6 2 20% 1 1  10% 80 4828 58% 12 25% 12  0 8 67% 8 0  80, 80, 70, 50, 40, 30, 20, 5%Investigation of Germline Transmission

Since the mice derived from three ES clones that are homologousrecombinants had favorable coat color chimeric rate, these chimeric micewere crossed with c57BL/6J female mice to investigate the germlinetransmission of the ES clone-derived chromosome. The following tablesummarizes the result.

TABLE 3 Total ES clone- No. of derived Percentage No. of offspringsAverage No. of mice Percentage Clone of chimera births ♂ ♀ babies born ♂♀ of germline 7 90% 5 13 14 5.4 12 11 85% 7 90% 4 19 11 7.5 16 9 83% 790% 3 10 19 9.7 0 0  0% 7 90% 6 14 19 5.5 13 18 94% 7 90% 0 0 0 0 0 0 0% 7 90% 5 10 22 6.4 3 13 50% Total of ES 44 51 clone-derived mice 76100%  2 4 5 4.5 2 1 33% 76 95% 5 10 16 5.2 0 3 12% Total of ES 2 4clone-derived mice 80 80% 1 2 3 5 0 0  0% 80 80% 0 0 0 0 0 0  0% 80 70%4 14 16 7.5 0 0  0%

Of six chimeric mice (all had a coat color chimeric rate of 90%) derivedfrom clone 7, four chimeric mice showed efficient germline transmission,and a total of 95 F1 mice could be obtained. Although the chimeric micederived from clone 80 did not exhibit germline transmission, chimericmice derived from clone 76 showed germline transmission, and thereforetwo independent lines of knock-in mice were established. Here in below,mice derived from these clones are designated as the 07 line and the 76line.

Gene Analysis of KI Mice

F1 mice can be judged based on coat color whether or not they areoffspring (agouti color) derived from the ES clone or offspring (black)derived from the host embryo. However, even the offspring derived fromthe ES clone have a probability of ½ of having the chromosome of theside that actually underwent homologous recombination. In order todifferentiate them, about 2 cm of the tail of the mice after ablactationwas harvested, and genomic DNA was extracted by KURABO's NA-1000.Analysis was performed on 100 ng of the genomic DNA using a PCR methodused in the above screening.

(4) Removal of the Neo Gene

In order to remove neo gene, the Cre expression vector (about 3 ng/μl)was injected into the pronucleus of eggs generated by in vitrofertilization of mature c57BL/6J oocytes with hTF KI sperm. At thistime, two vectors (pCAGGS-Cre-NLS and pCre-Pac) were used.pCAGGS-Cre-NLS is a pCAGGS vector in which the Cre gene has beeninserted at the C-terminal end of which a nuclear transfer signal (NLS)is added.

The pCAGGS vector is composed of the cytomegalovirus enhancer and thechicken β actin promoter, and it permits strong expression of theinserted gene in mammalian cells. On the other hand, pCre-Pac is avector in which Cre to which NLS is added at the N-terminal end isderived by the promoter of MC-1. Using these vectors, a procedure ofremoving the neo gene was carried out. The following table summarizesthe result.

TABLE 4 No. of No. of No. of No. of neo Name of surviving injected No.of No. of hTF KI deleted vector eggs eggs (%) transplantation offsprings(%) mouse mouse Efficiency pCAGGS-Cre 60  79 76% 56  8 14% 2 2 100%pCre-Pac 81 106 76% 80 17 21% 4 4 100%

As described in the above table, in either of the vectors the efficiencyof removing neo was equally 100%, but when Cre to which no NLS had beenadded was used, they were in a chimeric state in which neo was notcompletely extracted. The analysis was carried out using 5 μg of genomicDNA by a dot Southern blot method. Thus, it was found that the one forwhich the hTF gene was only detected using the neo gene and the hTF geneas probes was hTF KI mice in which neo has been removed, and the one forwhich detection was made with both probes was hTF KI mice in which neocould not be removed.

(5) Generation of hTF Homo Mice

hTF KI mice (-neo) obtained in the above were crossed with each other togenerate the homo mice (TF humanized mice) of hTF. In the gene analysisof these mice, the presence or absence of the hTFKI allele wasdetermined using the above PCR method, and using the primer set [mTF-5(forward) 5′-TTCACTCAAACCCACTGCGG-3′ (SEQ ID No. 11); mTF-H (reverse)5′-GCTACGCTACAGGAGCGATCG-3′ (SEQ ID No. 12], the presence or absence ofthe wild type allele was determined. PCR conditions were the same, butPCR reactions were carried out separately. In the completely humanizedhomo TF KI mice, bands are amplified for PCR alone for determining theKI allele.

Expression Analysis of hTF Mice

Tissues that express mTF were investigated using the Northern blotmethod. The mice used were four 8 week-old C57BL/6J ♂ mice. polyA+ RNAwas prepared from the brain, the lung, the thymus, the heart, the liver,the spleen, lymph nodes, the kidney, and the testis, and 5 μg of themwere used for the experiment (FIG. 2). The expression of mTF wasconfirmed in each tissue, and among them, expression was highest in thebrain and the lung. Therefore, the expression analysis of the preparedhTF KI mice was carried out on these tissues. The result is shown inFIG. 3.

The mice used are one month-old wild mice ♀ (+/+) (lanes 1 and 8) andthe same one month-old 07 line female (lanes 2-5, and 9-12). Among them,for hetero mice (KI/+) (lanes 2, 4, 9 and 11), homo mice (KI/KI) (lanes3, 5, 10, and 12), and those in which neo was further removed (−neo)(lanes 4, 5, 11 and 12), and those not removed (+neo) (lanes 2, 3, 9 and10), the expression of mTF was compared in the brain (lanes 1-5) and thelung (lanes 8-12). Also, the expression of hTF was compared between thelines on 4 month-old hetero mice, ♀, of the 07 line (lanes 6 and 13) andthe 76 line (7 and 14) and furthermore for the 07 line (lanes 2, 6, 9and 13) the expression of hTF was compared between ages.

These data confirmed that the knock-in vector caused homologousrecombination as designed giving mice that express hTF instead of mTF.Thus, the expression pattern of hTF in the established hTF KI mice isvery similar to that of the original mTF suggesting that knock-in micewere successfully established. There was no difference of expressionbetween the week-old ages or between the lines.

By crossing hTF KI mice (KI/KI, +neo) thus created with each other,propagative ability was examined. As a result, they exhibited apropagative ability of a similar degree to the wild type mice, and theproblem reported by Erlich et al. (Proc. Natl. Acad. Sci. USA 96:8138,1999) during gestation, that has been demonstrated in the doubletransgenic mice of the mTF knock-out mice and the hTF genome transgenicmice, was not observed in these mice.

TABLE 5 Performance of crossing in human TF knock-in mice Knock-in mouseFirst birth Second birth Third birth ♂No. 147 × ♀No. 115 1 ♀ 4 ♂ 3 ♀ 4 ♂4 ♀ 2 ♂ ♂No. 142 × ♀No. 111 3 ♀ 4 ♂ 5 ♀ 4 ♂ 3 ♀ 7 ♂ Wild type × Wildtype 1 ♀ 0 ♂ 3 ♀ 1 ♂ 3 ♀ 2 ♂ Wild type × Wild type 3 ♀ 4 ♂ 5 ♀ 4 ♂ 3 ♀ 7♂

EXAMPLE 2 Confirmation of TF Expression in Knock-In Mice

(1) Extraction of TF Protein

Brain acetone powders were prepared from five each of male and femalewild type mice C57BL/6J (CLEA Japan Inc.) and seven each of male andfemale 76 line human TF knock-in mice. After the cervical dislocation ofthe mice, craniotomy was performed to extract the whole brain, which wasquickly frozen in liquid nitrogen. To one g weight of the brain, 2 ml ofTBS (Tris-buffered-saline; Takara Shuzo) containing 1 mM PMSF(phenylmethylsulfonyl fluoride) was added, and the tissue washomogenized in a metal and Teflon homogenizer followed by centrifugationat 10,000×g, 4° C., for 1 hour to remove the supernatant.

TBS at an amount equal to the one described above was added to theprecipitate, which was stirred well, and centrifuged at 10,000×g, 4° C.,for 1 hour to remove the supernatant. To the precipitate obtained, 2 mlof ice-cold acetone per g of the brain was added and suspended well.After centrifuging at about 2,000×g, 4° C., for 20 minutes, thesupernatant was discarded. This was repeated and defatted. To theprecipitate obtained, nitrogen gas was injected to dryness, which wasprepared as a brain acetone powder.

400 μl of TBS containing 2% Triton X-100 was added to 10 mg of the brainacetone powder from each of the wild type mice, male and female, and thehuman TF knock-in mice, male and female, which were then subjected tosonication for 1 hour to be suspended. After recovering the supernatantby centrifugation (4° C., 15,000 rpm, 10 minutes), 1.2 ml of TBS wasadded for dilution, and then 1M MnCl₂ and 1M CaCl₂ were added to a finalconcentration of 1 mM. The above extract was added to about 50 μl of theConA Sepharose resin (Pharmacia Biotech) equilibrated with 0.5% TritonX-100/TBS (containing 1 mM MnCl₂ and 1 mM CaCl₂), and mixed by inventionat 4° C. overnight.

The resin was washed in 10 resin volumes of 0.5% Triton X-100/TBS(containing 1 mM MnCl₂ and 1 mM CaCl₂), and eluted with the SDS-PAGESample Buffer (Bio-Rad Laboratories), treated at 95° C. for 5 minutes,and centrifuged (25° C., 15,000 rpm, 1 minute) to collect thesupernatant, which was use as a sample. The CHO cell-derived solublehuman TF (see WO99/51743) and CHO cell-derived soluble mouse TF weremixed with an equal amount of the SDS-PAGE Sample Buffer (Bio-RadLaboratories) to prepare samples. Prestained Precision ProteinStandards™ (Bio-Rad Laboratories) was used as the molecular weightmarker.

(2) Detection of TF by Using Anti-TF Antibody

The extract from the brain acetone powder at 3 mg/lane equivalent andthe purified soluble TF at 100 ng/lane were electrophoresed on twosheets of 10% polyacrylamide gel (Tefco). It was electricallytransferred to the PVDF membrane (Millipore) in the Semidry typeprotting instrument, and protted in 1×PBS/Casein Blocker (Bio-RadLaboratories). One (for human TF detection) was reacted with 2 μg/ml ofanti-human TF antibody (American Diagnostica Inc., Cat. #4503), and theother (for mouse TF detection) was reacted with 2 μg/ml of anti-mouse TFantibody MFR-37 at room temperature for 1 hour.

After washing three times in PBS containing 0.05% Tween™ 20 for 5minutes, a secondary antibody diluted 1000-fold was reacted. Usingalkaline phosphatase-labelled goat anti-mouse IgG (H+L) (ZYMED, Cat.#62-6522) for the human TF detection system and alkalinephosphatase-labelled goat anti-rat Ig (BIOSOURCE, Cat. #ARI3405) for themouse TF detection, reaction was conducted at room temperature for 1hour. After washing three times in PBS containing 0.05% Tween™ 20 for 5minutes, color was developed with 1st Step NBT/BCIP (PIERCE). The resultis shown in FIG. 4. In FIG. 4, A represents a result of detection withanti-mouse TF antibody MFR-37, and B represents a result of detectionwith anti-human TF antibody ADI #4503. With anti-mouse TF antibody, TFwas detected only in the brain tissue of the wild type mouse, and withanti-human TF antibody, TF was detected only in the brain tissue of thehuman TF knock-in mouse.

(3) Detection of Human TF Activity

Using the brain acetone powder prepared as above, human TF activity wasdetected based on plasma coagulation activity. Human plasma used was thestandard human plasma (Dade Behring Marburg GmbH), and mouse plasma wascitrate-added plasma taken from the ICR mouse. The brain acetone powderwas suspended in TBS at 8 mg/ml, and mixed with equal amounts of 40, 4,0.4, 0 μg/ml of humanized anti-human TF antibody hATR-5 (ib2)(WO99/51743) diluted in TBS, and reacted at room temperature for 1 hour.An acetone powder-antibody mixture (50 μl/cup) was added to 100 μl/cupof plasma, and incubated at 37° C. for 3 minutes. 20 mM CaCl₂ (DadeBehring Marburg GmbH) was added thereto to start coagulation, andcoagulation time was measured using the Amelung KC-10A (Heinrich AmelungGmbH).

The results are shown in FIG. 5 and FIG. 6. FIG. 5 shows a result ofmouse plasma coagulation time in which the y-axis represents plasmacoagulation time, and each column represents the final concentration ofthe antibody. The coagulation activity of mouse plasma was detected at asimilar degree in the wild type and the human TF knock-in mouse. Withanti-human TF antibody, TF activity derived from the human TF knock-inmouse was only inhibited. FIG. 6 shows a result of human plasmacoagulation time, in which the y-axis represents plasma coagulationtime, and each column represents the final concentration of theantibody. The activity of wild type mouse TF was very weak compared tothat of the human TF knock-in mouse (Janson T L. et al., (1984)Haemostasis 14:440-444). With anti-human TF antibody, TF activityderived from the human TF knock-in mouse was only inhibited.

EXAMPLE 3 Creation of Improved Knock-In Mice

(1) Creation of Knock-In Mice

Mice in which the following two points have been improved compared tothe mice created in Example 1 were established in a manner similar toExample 1.

Modification 1

The initiation codon ATG which is a protein translation initiation codonof the mouse TF gene was converted to CTG to introduce mutation in themouse TF gene.

Modification 2

A sequence following the termination codon (codon TAA corresponding to897-899 of SEQ ID NO: 7) of the human TF gene was modified to thesequence of 3′ UTR of mouse TF.

The 3′ UTR sequence of the mouse TF gene used is the 3′ UTR region (fromthe base at position 989 to the last base (TAGAGGAAA-tgactccg, SEQ IDNO: 19)) of the sequence of the mouse genomic sequence described inArteriosclerosis and Thrombosis 12(4):474 (1992)).

Specifically, using the genome of C57BL/6J mice as template, and thefollowing PCR primers, PCR cloning was performed. PCR was performed in areaction system of a total of 50 μl using 100 ng of template genomic DNAand TaKaRa LA Taq. The PCR condition was 95° C. for 1 minute, 35 cycles(95° C. for 0.5 min, 62° C. for 0.5 min, 72° C. for 1 min), and finally72° C. for 7 minutes, and then reaction was terminated at 4° C.

FmTF3UTR: 5′-TCATCCTCCTGTCCATATCTCTGTGC-3′ (SEQ ID NO: 13) RmTF3UTR:5′-CGGAGTCACCTAATGTGAAAACCAAG-3′ (SEQ ID NO: 14)

The amplified fragment was subjected to TA cloning (Promega) to confirmthe sequence, and was confirmed to be identical with the sequencedescribed in the above article.

mTF 3′ UTR of C57BL/6J mice obtained was added to the cording region ofhTF (hTF CDS) by assemble PCR. To assemble PCR, Pyrobest (TaKaRa) wasused.

Each of hTF cording region and mTF 3′ UTR was amplified by PCR usingprimers hTFassem-mTFhTF, mTFassem-hTFmTF (hTFassem: 5′-GGA TCC TCG AGGCCA CCA TGG AGA CCC CTG-3′ (SEQ ID No. 15) (Xho I and BamHI sites wereadded), mTFassem: 5′-TCT AGA CTC GAG CGG AGT CAC CTA ATG TGA-3′ (SEQ IDNo. 16) (Xho I and Xba I sites were added), hTFmTF: 5′-ACT CCC CAC TGAATG TTT CAT AAA GGA AAG GCT GAA GCG C-3′ (SEQ ID No. 17), mTFhTF: 5′-GCGCTT CAG CCT TTC CTT TAT GAA ACA TTC AGT GGG GAG T-3′ (SEQ ID No. 18),and the fragments obtained were purified. PCR was conducted at thecondition of 95° C. for 1 minute, 30 cycles (95° C. for 0.5 min, 60° C.for 0.5 min, 72° C. for 1 min), 72° C. for 7 minutes and 4° C.

The purified hTF cording region and mTF 3′ UTR fragment were mixed, andsubjected to assemble PCR at the following condition. After two cycles(94° C. for 2 min, 58° C. for 2 min, 72° C. for 2 min) in the absence ofprimers, hTFassem and mTFassem primers were added, and 30 cycles (95° C.for 0.5 min, 58° C. for 0.5 min, 72° C. for 21 min), and finally atreatment of 72° C. for 7 minutes was carried out, and then reaction wasterminated at 4° C.

Using the DNA obtained, a knock-in mouse was created in a manner similarto Example 1.

(2) Expression Analysis of hTF

In a manner similar to Example 1, the expression of TF in the brain wasanalyzed by Northern blot method.

For expression analysis, there were used those in which the human TFgene (knocked-in gene) is present in only one of the chromosomes and theother has been conserved (wild type) in both of the unimproved knock-inmice (the mouse in Example 1) and the improved knock-in mice.Accordingly, both of the unimproved knock-in mice and the improvedknock-in mice express both of human TF and mouse TF.

Thus, when a human gene inserted into the mouse is highly expressed, theuse of mouse 3′ UTR instead of human 3′ UTR is a very effective method.

This method is believed to be applicable not only to the mouse but alsoto the creation of other knock-in non-human animals, and by adding the3′ UTR derived from the knock-in non-human animal to the gene to beinserted, the inserted gene can be controlled to be expressed at a highlevel.

EXAMPLE 4 Arterial Occlusion Model

Using the human TF knock-in mouse the 76 line (♂, 8-20 week-old), anarterial thrombosis model was created. Mice were anesthetized with theadministration of pentobarbital (50 mg/kg, i.p.), and the mice wereretained at the dorsal position, and the rectal temperature measuringprobe was mounted thereon. An incision was made in the neck to removethe left common carotid artery, from which nerves running in parallelwith the artery were peeled to isolate the artery from the othertissues. A humanized anti-human TF antibody hATR-5 (ib2) (WO99/51743)(30 mg/kg) or the solvent therefor (20 mM sodium acetate buffer, pH 6.0,containing 150 mM NaCl) were intravenously given (50 μl/10 g B.W.)through the tail vein or the right common cervical vein. A soft cuffprobe 0.5 mm, 20 MHz, (Crystal Biotech) connected to the Pulsed DopplerFlow/Dimension System, Model SAGE3 (Triton Technology, Inc.) was mountedto the artery, and monitored on the chart recorder LINEARCORDER mark VIIWR3101 (Graphtec). After the passage of 5 minutes or more after the drugadministration, a stimulus was given to the artery at the condition ofrectal temperature of 36-37° C. A glycerin solution containing 40 w/v %FeCl₃.6H2O (FeG) was attached drop by drop to the surface of the bloodvessel on the heart side from the probe mounting site of the commoncarotid artery for stimulation. After stimulation for 2 minutes, FeG wasremoved, and while monitoring the blood flow shown on the chart, thecanalization of the artery was monitored. The observation lasted for 30minutes after the start of stimulation, and the canalization time wasdetermined and the patency rate was calculated.

The administration of hATR-5 (ib2) significantly prolonged the patencyduration, and the patency rate increased significantly, and thussuppressed arterial obstruction significantly.

Patency Patency rate Group duration (min) (%) Solvent administrationgroup 15.7 ± 1.5 52.4 ± 5.1 (n = 19) hATR-5 (ib2) administration 20.5 ±1.2 68.4 ± 4.1 group (n = 19) P value 0.020 0.020

EXAMPLE 5 Creation of Anti-Mouse TF Antibody Using the Human TF Knock-InMice

Mouse TF was emulsified by mixing with an equal volume of Freund'scomplete adjuvant (Difco Laboratories) in a glass syringe. As mouse TF,CHO cell-derived soluble TF (smTF) was used. By subcutaneous injectionof 10 μg/mouse, three mice each of male knock-in 07 line and 76 linewere immunized. Two weeks later, smTF was emulsified by mixing with anequal amount of Freund's incomplete adjuvant (Difco Laboratories)(smTF-FIA) in a glass syringe, 10 μg/mouse of which was subcutaneouslyinjected for immunization. Thereafter, smTF-FIA was subcutaneously givenevery week for immunization. 10 μg/mouse equivalent of smTF was injectedfor the second to fourth weeks, 30 μg/mouse equivalent for the fifthweek, and 20 μg/mouse equivalent for the sixth and seventh weeks, forimmunization.

Blood was taken from the orbit immediately before each immunization,serum was separated and the antibody titer of anti-mouse TF antibody wasdetermined by ELISA. Blood drawing after the seventh immunization wasperformed four days after the immunization. The procedure for ELISA isshown below.

smTF was diluted to 1 μg/ml in the immobilization buffer, dispensed intoa 96-well microwell plate (Maxisorp; Nunc) at 100 μl/well to immobilizesmTF. As the immobilization buffer, sodium bicarbonate solution (pH 9.6)containing 0.02 w/v % NaN₃ was used. After washing three times with awash buffer at 300 μl/well, it was blocked with a blocking buffer at 200μl/well. The wash buffer used was PBS(−) containing 0.05 vol % of Tween™20, and the blocking buffer used was 50 mM Tris-HCl buffer (pH 8.1)containing 0.15 M NaCl, 1 mM MgCl₂, 0.05 vol % of Tween™ 20, 0.02 w/v %NaN₃, and 1 w/v % bovine serum albumin. After discarding the blockingbuffer, antiserum diluted as appropriate with the blocking buffer wasreacted at 100 μl/well (room temperature, 1 hour). After washing threetimes with the wash buffer at 300 μl/well, AP-Goat Anti-Mouse IgG (H+L)(ZYMED Laboratories) diluted 1,000-fold in the blocking buffer wasreacted at 100 μl/well (room temperature, 2 hours). After washing fivetimes with the wash buffer at 300 μl/well, an alkaline phosphatasesubstrate solution was dispensed at 100 μl/well to develop color at roomtemperature, absorbance was measured at 405 nm (reference 655 nm).Absorbance was measured using the Model 3550 Microplate Reader(Bio-Rad). The solution was 50 mM sodium bicarbonate solution (pH 9.8)containing 1 mg/ml SIGMA™ 104 and 10 mM MgCl₂.

As a result, antibody titer to mouse TF increased and antiserum wasobtained.

The result are shown in FIGS. 7-12.

EXAMPLE 6 Creation of LPS-Induced DIC Model

Sepsis was induced by administering LPS to each of the wild type mouseand the human TF knock-in mouse. The wild type mouse used is C57BL/6J(CLEA Japan, Inc.), female, 15 weeks old, and the human TF knock-inmouse used is the 76 line, female, 14 weeks old. LPS used is derivedfrom Escherichia coli (serotype 0111: B4) (SIGMA-Aldrich Co.). LPS wasdissolved in physiological saline, and 0.5 mg/kg was given to the wildtype mouse, and 0, 1, 5, 25 mg/kg was given to the hTF knock-in mouseintraperitoneally (0.1 ml/10 g B.W.). Six hours after theadministration, blood was taken from the abdominal portion of vena underether anesthesia. At this time, 3.8% citric acid was added to a finalvolume of 1/10 as an anticoagulant. Platelet count was measured using anautomated blood cell counter F-800 (Sysmex).

Platelet count decreased confirming that the symptom of DIC developed.

LPS dose hTFKI mice C57BL/6J mice 0 mg/kg 103.1 ± 19.3 (5)  111.4 ± 5.1(5) 1 mg/kg 80.1 ± 15.5 (5) — 5 mg/kg 78.1 ± 12.1 (5)  63.4 ± 8.5 (4) 25mg/kg   70.6 ± 8.5 (4) — ( ) indicates the number of animals.

REFERENCE EXAMPLE 1 Creation of Anti-Mouse TF Antibody

(1) Preparation of Antigen

A gene encoding the recombinant soluble mouse tissue factor protein(smTF) in which a sequence of amino acids 1-29 at the N terminal end ofthe amino acids 1-251 (Hartzell S. et al. (1989) Mol. Cell. Biol.9:2567-2573) of mouse tissue factor (mouse TF) was replaced withmethionine for E. coli expression and a FLAG peptide (DYKDDDDK, SEQ IDNO: 20) was added to the C terminal, and it was inserted into an E. coliexpression vector containing T7 promoter, which was introduced into E.coli strain BL21.

IPTG at a final concentration of 1 mM was added to the culture oftransformed E. coli to induce the expression of smTF, E. coli wascollected, and the lysate was analyzed on SDS-PAGE with a result thatsmTF was expressed as a protein of molecular weight about 25 kDa. Thetransformed E. coli was cultured at 2 L, and when absorbance at 600 nmreached 0.2, IPTG was added at a final concentration of 1 mM, which wasfurther cultured for 5 hours to harvest the cells. The cells collectedwere stored frozen at −80° C.

After the cells were disrupted by sonication, the precipitate after celldisruption was washed three times with 4% Triton X-100 and 2 M urea, wasfurther washed twice with MilliQ water, and then was solubilized in 100ml of 8 M urea/10 mM dithiothreitol/50 mM Tris-HCl buffer (pH 8.5). Itwas centrifuged at 25,000×G for 20 minutes to remove the insolublesubstances and to recover soluble substances, which were designated assmTF(U). In order to allow the smTF(U) to refold, 50 ml of smTF(U) wasdialyzed against 5 mM reduced glutathione/1 mM oxidized glutathione/20mM Tris-HCl buffer (pH 7.4), and two hours and six hours later thedialysis external solution was changed for further dialysis for 16hours.

A solution obtained by centrifuging the dialysis internal solution at25,000×G for 20 minutes was desalted with 20 mM Tris-HCl buffer (pH 7.4)to remove glutathione, which was then added to the Resource Q6ml column(Pharmacia Biotech) equilibrated with 20 mM Tris-HCl buffer (pH 7.4),and washed in 2 column volumes of the same buffer. In the 20 mM Tris-HClbuffer (pH 7.4), NaCl concentration was linearly increased from 0 M to0.3 M to elute soluble mouse TF. The soluble mouse TF fraction obtainedwas designated as smTF(R).

(2) Immunization and Preparation of Hybridoma

Recombinant soluble mouse tissue factor (TF) was emulsified(oil-in-water type) by mixing with an equal amount of Freund's completeadjuvant (Difco Laboratories) in a glass syringe for about 30 minutes.As TF, smTF(R) at 50 μg/rat and smTF(U) at 100 μg/rat weresubcutaneously inoculated to the lamb of 6 week-old male Wistar rats(Nippon Charles River).

As a booster, Freund's incomplete adjuvant (Difco Laboratories) insteadof Freund's complete adjuvant was used in a similar manner to prepare anemulsion, which was administered on day 15 and 22 after the initialimmunization at 50 μg/rat for smTF(R) and at 100 μg/rat for smTF(U), andfurthermore on day 29, 43, and 50 after the initial immunization,smTF(R) at 25 μg/rat and smTF(U) at 50 μg/rat were subcutaneouslyinoculated to the lamb of the rats. Using the immunogen described belowas antigen in ELISA, a sufficient increase in antibody titer wasconfirmed, and then as a final immunization on day 57 after the initialimmunization, smTF(R) at 50 μg/rat and smTF(U) at 100 μg/rat wereadministered via the tail vein.

Three days after the final immunization, spleen cells were asepticallyprepared from the rats, and 1.85×10⁸ spleen cells prepared and 1.85×10⁷mouse myeloma cells P3/X63-Ag8.U1 (hereinafter P3U1) were mixed, and thecells were fused according to a standard method (Harlow, E. and Lane, D.(1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory)using polyethylene glycol 1500 (BOEHRINGER MANNHEIM). The fused cellswere suspended in a RPMI 1640 medium (GIBCO BRM) containing 10% bovinefetal serum (INTERGEN) (hereinafter RPMI medium), and plated to 96-wellmicro plate at a P3U1 density of 5×10⁴ cells/100 μl/well, and cultured.

The number of 96-well micro plates onto which the cells are plated wasfour plates per rat (the number of wells, 352). On the next day, RPMImedium containing 2% BM-condimed H1 (BOEHRINGER MANNHEIM) and HAT (GIBCOBRL) (hereinafter, HAT medium) was added at 100 μl/well, and on day 2,3, and 5 after cell fusion, the half amount was replaced with the HATmedium for HAT selection.

On day 9 after cell fusion, the antibody titer of each well wasdetermined by cell ELISA as a primary screening. Cells from wells thatexhibited high absorbance were subcultured to 12 well plates for asecondary screening.

Four days after the primary screening, as a secondary screening,antibody titer by cell ELISA and by ELISA using as antigen smTF(U) andsmTF expressed in COS cells (smTF(cos)) and the inhibitory activity ofFactor Xa production were determined. For the wells in which theinhibitory activity of both Factor Xa production and Factor X bindingwere observed, typing of antibodies were performed.

Hybridomas selected in the secondary screening were cloned by repeatingtwice limiting dilution, and conditioned in an RPMI 1640 mediumcontaining 2% BM-codimed H1. After confirming productivity of antibody,this was considered to be the completion of establishment of hybridomas.

Finally, six clones of antibody (MFR-36, MFR-37, MFR-40, MFR-66 (IgG2a),MFR-25, MFR-51 (IgM)) having both of the inhibitory activity of humanFactor Xa production and the inhibitory activity of human Factor Xbinding were established.

(3) Preparation of Ascites and Purification of Antibody

Preparation of ascites of the hybridoma established was carried outaccording to a standard method. Thus, 1×10⁶ hybridoma (MFR-37) cellsthat had been subcultured in vitro were intraperitoneally transplantedto male BALB/c nude mouse, BALB/c-nu/nu (Japan Charles River) that hadintraperitoneally received mineral oil twice. The ascites was recoveredfrom the mouse that exhibited a bloated abdomen 1-2 weeks after thetransplantation.

Purification of antibody (MFR-37) from the ascites was performed outusing a combination of SP Sepharose Fast Flow column chromatography andProtein G Sepharose 6 FF column chromatography. Thus, it was diluted10-fold with 20 mM sodium acetate buffer (pH 5.0), allowed to adsorb toa SP Sepharose Fast Flow column (50 mm ID×130 mmH), washed with 20 mMsodium acetate buffer (pH 5.0), and eluted in 20 mM sodium acetatebuffer (pH 6.0) containing 500 mM sodium chloride. The eluted fractionwas adjusted to about pH 6 with 1 M Tris solution, and was allowed toadsorb to a Protein G Sepharose 6 FF column (16 mm ID×125 mmH).

After washing with about 10 column volumes of 20 mM sodium acetatebuffer (pH 6.0), it was eluted with 50 mM acetic acid. After the elutedfraction was diluted two-fold in distilled water, pH was adjusted to 5.0with 1 M Tris solution, and was allowed to adsorb to an SP Sepharose XLcolumn (10 mm ID×100 mmH). After washing with about 10 column volumes of20 mM sodium acetate buffer (pH 5.0), it was eluted with 20 mM sodiumacetate buffer (pH 6.0) containing 200 mM sodium chloride to preparepurified antibody.

REFERENCE EXAMPLE 2 Preparation of CHO Cell-Derived Soluble Mouse TF

A gene encoding protein in which a FLAG peptide (DYKDDDDK, SEQ ID NO:20) was added to the C terminal of the amino acids No. 1-251 (HartzellS. et al. (1989) Mol. Cell. Biol. 9: 2567-2573) of TF was inserted to anexpression vector for mammals containing the DHFR expression gene, andintroduced into CHO cells. Methotrexate was used to amplify expressionand the soluble mouse TF-producing CHO cells were established.

The cells were cultured in a serum-free CHO-S-SFM II (GIBCO BRL) toobtain a culture supernatant containing soluble mouse TF. Two volumes of20 mM Tris-HCl buffer (pH 8.5) were added to the culture supernatant,and diluted three-fold. It was added to a Q-Sepharose Fast Flow column(200 ml, Pharmacia Biotech) equilibrated with 20 mM Tris-HCl (pH 8.5),and washed with three column volumes of the same buffer. In 20 mMTris-HCl buffer (pH 8.5), NaCl concentration was linearly increased from0 M to 1 M to elute soluble mouse TF.

Ammonium sulfate was added to the soluble mouse TF thus obtained to a30% saturation, and after centrifugation contaminating proteins wereprecipitated. The supernatant was recovered, to which ammonium sulfatewas further added to a 50% saturation, and after centrifuge,contaminating proteins were precipitated. The supernatant was applied onButyl-TOYOPEARL (21.5 ml, TOSOH), and washed with three column volumesof 50 mM Tris-HCl buffer (pH 6.8) containing 1.8 M ammonium sulfate. In50 mM Tris-HCl buffer (pH 6.8), the ammonium sulfate concentration waslinearly decreased from 1.8 M to 0 M to elute soluble mouse TF.

Peak fractions containing soluble mouse TF were concentrated byCentri-Prep 10 (Amicon). The concentrate was applied on the TSKgel G3000SWG column (21.5×600 mm, TOSOH) equilibrated with 20 mM sodium phosphatebuffer (pH 7.0) containing 150 mM NaCl, and the peak fraction of solublemouse TF was collected. This was sterilized with a 0.22 μm membranefilter to prepare the CHO cell-derived soluble mouse TF. Theconcentration of the sample was calculated using the molar extinctioncoefficient of the sample of ε=39,670 and molecular weight of 45,000.

1. A human tissue factor (TF) homozygous mouse whose genome comprises ahuman tissue factor gene that has been inserted upstream of thetranslation initiation codon for the mouse TF gene and is operablylinked to the endogenous mouse TF promoter, wherein the mouse produceshuman TF without producing mouse TF, and the transgene is inserted in a129 SvEv C57B1/6J genetic background, and wherein the mouse exhibits aphenotype of a propagative ability similar to that of a wild type mouse,and inhibited blood coagulation activity by human TF upon administrationof an anti-human TF antibody.
 2. The human tissue factor homozygousmouse according to claim 1 wherein the human TF-encoding gene comprisesan AU-rich response element (ARE) or a polyA additional signal at the3′-end thereof.
 3. The human tissue factor homozygous mouse according toclaim 2 wherein the human TF-encoding gene comprises an AU-rich responseelement (ARE) and a polyA additional signal at the 3′-end thereof.